Abstract
A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.
Highlights
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related mortality worldwide [1,2,3]
This is related to high drug resistance to six anticancer drugs when HCC cells are cultured in the aggregated spheroid model (ASM), suggesting the ASM is superior to the conventional 2D cell culture-based drug testing assay
We have successfully optimized 3D cell culture methods and created spheroids by aggregating cells in Matrigel attached on the surface of a 96-pillar plate
Summary
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related mortality worldwide [1,2,3]. HepG2 and Hep3B had E-cadherin values of 6.88 and 3.21, respectively (Figure 3A,B) These amounts were greater than those found in the 2D and CSM cell culture models. The expression levels of the ECM component (Fibronectin) and structure of tight junctions (ZO-1, Occludin) were measured by western blot analysis to understand the differences of the drug response in each cell culture model. The expression level of ZO-1 was measured as 1.81 and 3.44 in HepG2 and Hep3B, respectively These levels were greater than those in the corresponding 2D and CSM cell culture model counterparts, as shown in ecules 2021, 26, x FOR PEER REVIEW. We confirmed that the epithelial characteristics of HCC cells were better maintained, and the expression of Fibronectin and tight junction proteins related to drug resistance was higher in the ASM. This is related to high drug resistance to six anticancer drugs when HCC cells are cultured in the ASM, suggesting the ASM is superior to the conventional 2D cell culture-based drug testing assay
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
